This project proposes to determine the role of cis-acting sequences in the control of gene expression and episome stability in Entamoeba histolytica. E. histolytica is an extracellular protozoan parasite responsible for amebic colitis and liver abscess, diseases that afflict upwards of 50 million people annually in the developing world. Progress in diagnosis, treatment, and immunoprophylaxis have all been hindered by the inability to apply genetic analysis to E. histolytica. There is no recognized sexual cycle, and until recently no mechanism to introduce and express foreign DNA in this (or any other) enteric parasite. The development, by our laboratory and others, of techniques for transient and stable transfection makes possible experimental investigation into important and unstudied questions of the biology and virulence of E. histolytica. In this project we propose to begin the study of the regulation of gene expression by dissecting the upstream and downstream regulatory regions of the developmentally regulated lectin hgl1 gene, and investigate the role of cis-acting sequences in maintaining the parasite's extrachromosomal rDNA episome. Preliminary results have demonstrated the importance of both 5' and 3' flanking hgl1 gene sequences for reporter expression. Deletional and mutational analyses have further defined 5' flanking sequences with positive and negative effects on reporter gene expression. Insight into the maintenance of the rDNA episome has been achieved with the observation that ligation of a region of tandem repeats from the rDNA episome into transfection plasmids stabilizes them in the absence of selection. Three specific aims are proposed: (A) Optimize transient and stable transfection in E. histolytica - including determination of transfection efficiency of episomal transfection, and development of techniques for gene replacement via homologous recombination or restriction-enzyme mediated integration; (B) Identify and characterize the function of sequences from the rDNA episome which confer episome stability including deletional and mutational analysis of the cis-acting sequence and definition of its function as either a segregation sequence or replication origin; and (C) Analysis of cis-acting sequences(DNA-binding proteins controlling expression of the lectin hgl1 gene - including analysis of endogenous stage-specific hgl1 transcription, deletional and mutational analyses of 5' and 3' flanking DNA of hgl1, and identification/purification of sequence-specific DNA binding proteins recognizing these cis-acting sequences. Successful completion of the proposed studies will give insight into the novel regulatory elements controlling gene expression and episome stability in E. histolytica, and will establish the basis for genetic approaches to the study of E. histolytica cell biology and pathogenesis.